Bone protein extraction without demineralization

ABSTRACT

Embodiments of the invention relate generally to protein extraction and, more generally, to bone protein extraction methods that do not require demineralization. In one embodiment, the invention provides a method comprising: mixing a bone sample and a quantity of an extraction buffer comprising: ammonium phosphate dibasic; or ammonium phosphate dibasic and ammonium bicarbonate; or ammonium phosphate dibasic, ammonium bicarbonate, and guanidine HCl; or sodium phosphate dibasic and sodium bicarbonate; or sodium phosphate dibasic, sodium bicarbonate, and guanidine HCl; or potassium phosphate dibasic and potassium bicarbonate; or potassium phosphate dibasic, potassium bicarbonate, and guanidine HCl; and incubating the bone sample/extraction buffer mixture.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/533,652, filed Jun. 6, 2017, which is a national stage filing ofInternational Patent Application No. PCT/US2015/065239, filed Dec. 11,2015, which claims the benefit of U.S. Provisional Application Ser. No.62/090,612, filed Dec. 11, 2014, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no. R01AR049635 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Bone is a mineralized tissue. As such, extracting proteins from bonetypically requires a demineralization step. Such steps are slow (manytake days to weeks to perform) and negatively impact subsequentextraction of the proteins. Specifically, demineralization breaks downproteins by hydrolysis. This reduces not only the total protein contentrecovered from bone samples, but also the types of proteins recovered.For example, most demineralization-based extraction methods recover onlyabout 1% of the original bone mass as protein, most of which is composedof collagen I proteins.

Extraction methods that do not involve demineralization have beendeveloped. These have had very low total protein yields, however,typically 3 mg or less of protein per gram of bone.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a method comprising: mixing abone sample and a quantity of an extraction buffer comprising: ammoniumphosphate dibasic; or ammonium phosphate dibasic and ammoniumbicarbonate; or ammonium phosphate dibasic, ammonium bicarbonate, andguanidine HCl; or sodium phosphate dibasic and sodium bicarbonate; orsodium phosphate dibasic, sodium bicarbonate, and guanidine HCl; orpotassium phosphate dibasic and potassium bicarbonate; or potassiumphosphate dibasic, potassium bicarbonate, and guanidine HCl; andincubating the bone sample/extraction buffer mixture.

In another embodiment, the invention provides an extraction buffercomprising: ammonium phosphate dibasic; or ammonium phosphate dibasicand ammonium bicarbonate; or ammonium phosphate dibasic, ammoniumbicarbonate, and guanidine HCl; or sodium phosphate dibasic and sodiumbicarbonate; or sodium phosphate dibasic, sodium bicarbonate, andguanidine HCl; or potassium phosphate dibasic and potassium bicarbonate;or potassium phosphate dibasic, potassium bicarbonate, and guanidineHCl.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of this invention will be more readilyunderstood from the following detailed description of the variousaspects of the invention taken in conjunction with the accompanyingdrawings that depict various embodiments of the invention, in which:

FIG. 1 shows protein yields from bone samples of common origin usingdifferent extraction buffers of the invention at four-, eight-, and24-hour intervals;

FIG. 2 shows protein yields from bone samples of common origin usingdifferent extraction buffers of the invention incubated at roomtemperature and 75° C.;

FIG. 3 shows average protein yields for a number of bone samples usingdifferent extraction buffers of the invention incubated at 75° C.;

FIG. 4 shows protein yields for a number of bone samples using anextraction buffer comprising 400 mM ammonium phosphate dibasic/200 mMammonium bicarbonate incubated at 75° C.;

FIG. 5 shows protein yields of extraction buffers according to theinvention with varying buffer volumes and phosphate concentrations;

FIG. 6 shows a plot of protein concentration as a function of bonesample mass using extraction buffers according to the invention;

FIG. 7 shows protein yields for various extraction buffers withdifferent cations according to embodiments of the invention; and

FIG. 8 shows protein yields for extraction buffers includingphenacylthiazolium bromide (PTB) according to embodiments of theinvention.

It is noted that the drawings of the invention are not to scale. Thedrawings are intended to depict only typical aspects of the invention,and therefore should not be considered as limiting the scope of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

In hydroxyapatite chromatography, proteins are eluted from thehydroxyapatite column with increasing phosphate concentration. Becausebone is a composite of hydroxyapatite and protein, the use of higherconcentration phosphate buffers similar to the final concentrations usedin hydroxyapatite chromatography in bone protein extraction methodsallows higher and more complete protein yields than has been achievableusing other extraction methods that do not include a demineralizationstep.

Specifically, extraction buffers comprising ammonium phosphate dibasic(typically at a concentration between about 400 mM and about 1 M) orammonium phosphate dibasic and ammonium bicarbonate (typically at aconcentration of about 200 mM) were employed according to someembodiments of the invention. In other embodiments, extraction bufferscomprising ammonium phosphate dibasic, ammonium bicarbonate, andguanidine HCl (at a concentration of about 4M) were employed.

The example below illustrates illustrative methods and extractionbuffers according to embodiments of the invention, as well as theresults of the mass spectrometry of proteins extracted using thesemethods and buffers. One skilled in the art will recognize, of course,that various modifications of the methods and extraction buffersdescribed are possible and such modifications are within the scope ofthe invention.

Tibial cortical bone samples were sampled from seven Caucasian cadavers(23F, 25M, 48M, 56M, 79M, 81F, 82M). All samples were previouslydiagnosed to be free from metabolic bone diseases, HIV, and hepatitis B.No live human subjects were involved in this research study.

Utilizing phosphate elution principles from hydroxyapatitechromatography, extraction buffers comprising either 400 mM ammoniumphosphate dibasic or 400 mM ammonium phosphate dibasic/200 mM ammoniumbicarbonate were prepared. To determine differences between the twoextraction solutions, a number of initial tests were performed on boneobtained from a 48-year-old male. Bone samples (100 mg each; fragmentedto ˜1 mm³) were extracted in 600 μL of solutions of 400 mM ammoniumphosphate dibasic or 400 mM ammonium phosphate dibasic/200 mM ammoniumbicarbonate after homogenization using stainless steel beads in a BulletBlender. Because this is a tube based homogenization method, particlesize was not measured. Aliquots were taken at 4, 8, and 24 hrs toevaluate the amount of time necessary to extract protein for eachsolution.

After initial set of tests, the extraction was repeated on ˜50 mg ofbone with 400 mM ammonium phosphate, 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate, 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate/4M guanidine HCl (GuHCl) for a fixedperiod of 24 hours only. Temperature was varied at 4° C., roomtemperature, or 75° C. to determine the effects of temperature onextraction. Finally, an additional ˜50 mg of bone was extracted at 75°C. with 200 mM ammonium bicarbonate for 24 hrs for comparison to theammonium phosphate extractions. The 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate/4M GuHCl extraction was only testedat 75° C.

After establishing the method with the highest yields, ˜50 mg of bonefrom other cadaveric donors were extracted using the 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate extraction for 24 hrs at75° C. Protein concentration was determined using Coomassie (Bradford)Assay Kit (Thermo-Scientific) with BSA as a protein standard, and allsamples were desalted using micro dialysis (3500 MWCO regeneratedcellulose; Fisher Scientific) against nanopure water for 4 days.

To evaluate if proteolysis occurs during the 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate or the 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate/4M GuHCl extraction process,additional 50 mg aliquots of the 48M samples were homogenized with theinclusion of 10 μg/mL Halt™ Protease Inhibitor (Thermo-Scientific) andincubated for 24 hr at 75° C.

The 400 mM ammonium phosphate dibasic extraction and all 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate samples were reduced with10 mM dithiothreitol for 1 hour at room temperature followed byalkylation using 30 mM iodoacetamide for 1 hour in the dark. Proteinswere digested overnight with Trypsin Gold (Promega) at 37° C. (1:100trypsin:protein). Peptide samples were prepared for mass spectrometryusing a C18 stage tip. After binding to the C18 disk, samples werewashed with 50 μL of 0.1% formic acid and eluted using 20 μL of 80%acetonitrile, 0.1% formic acid. Samples were partially dried in air toremove excess acetonitrile and resuspended to a final volume of 15 L in0.1% formic acid. Prepared peptides were separated using an Agilent 1200Series HPLC with a Thermo Scientific BioBasic C18 (2.1 mm ID, 100 mmcolumn length, 5 m particle size) for 75 minutes using either of thefollowing gradients: 1) 2% B 0-5 min, 30% B 5-15 min, 60% B 15-60 min,95% B 60.01-64 min, 2% B 64.01-75 min or 2) 2% B 0-5 min, 30% B 5-35min, 60% B 35-60 min, 95% B 60.01-64 min, 2% B 64.01-75 min where A is0.1% formic acid and B is 100 acetonitrile, 0.1% formic acid. Elutedpeptides were characterized on a LTQ-Orbitrap XL (Thermo Scientific).The top 2 peaks were fragmented using either collision induceddissociation (CID) or higher energy collisional dissociation (HCD) inthe Orbitrap or the top 5 peaks were fragmented with CID and analyzed inthe ion trap. All samples were analyzed by mass spectrometry intriplicate.

Peak lists (mgf) were created in MassMatrix Mass Spectrometric FileConversion Tools v. 3.2. Peak lists were searched against Swissprot anda decoy database using Mascot 2.3 (Matrix Science). The followingparameters were set for each search: taxonomy was set to Homo sapiens;enzyme=trypsin; up to 3 missed cleavages; variable modifications:carbamidomethyl (C), deamidation (NQ), carboxy (E), oxidation (MKP);static modifications: none; peptide tolerance=10 ppm; fragmenttolerance=0.5 Da; and peptide charge=2+, 3+, 4+. Peptide results werefiltered using Percolator at p<0.05. Peptides with nonsensicalpost-translational modifications (e.g., carboxyglutamic acid (Gla) onnon-Gla containing proteins) were filtered by hand.

To evaluate the differences in protein yield between extraction types,one-way ANOVA was performed in SigmaStat for Windows 2.03 (SPSS Inc.).Significance was set at p<0.05.

The 400 mM ammonium phosphate dibasic/200 mM ammonium bicarbonate had asignificantly greater yield than 400 mM ammonium phosphate dibasic alonefor all times (p<0.001), as shown in FIG. 1 . In the figures, * isp<0.05, ** is p<0.01, and *** is p<0.0001. No variation in yield wasobserved between times.

Temperature change resulted in a significant increase (p<0.001) inprotein concentration for both the 400 mM ammonium phosphate dibasic and400 mM ammonium phosphate dibasic/200 mM ammonium bicarbonate solutions(FIG. 2 ). Very little yield (1.05±0.16 mg of protein/g bone) wasdetected after extraction with 200 mM ammonium bicarbonate at 75° C.(FIG. 3 ); much less than either ammonium phosphate extraction(p<0.001). The highest yield was obtained with the 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate/4M GuHCl extraction(p<0.001). Extractions from bones obtained from the other cadavericdonors resulted in yields between 3.53±0.42 and 7.79±0.23 mg protein pergram of bone (FIG. 4 ).

For both extractions from the 48-year-old donor, peptides from collagenI were the most abundantly detected. Osteocalcin and ceruloplasmin wereonly detected in the 400 mM ammonium phosphate dibasic/200 mM ammoniumbicarbonate extractions using the top 2 CID method, and actin, serumalbumin, and apolipoprotein A-1 were only detected in the 400 mMammonium phosphate dibasic extraction also only using the top 2 CIDmethod.

Hemoglobin, vimentin, and fibrinogen gamma chain peptides were detectedfor both 400 mM ammonium phosphate dibasic and 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate. Osteocalcin was detected in the 400mM ammonium phosphate dibasic extraction when using the top 5 CIDfragmentation method.

While as few as four hours of extraction was found sufficient for the400 mM ammonium phosphate dibasic/200 mM ammonium bicarbonate method, 24hours was used for the individual ages to maximize the amount and typesof protein extracted for mass spectrometry. In all samples for the 400mM ammonium phosphate dibasic/200 mM ammonium bicarbonate extractionfragmented using the top 5 method, collagen I alpha-2 and alpha-1 wereconsistently the most abundant and second most abundant protein chainsdetected, respectively. Osteocalcin was detected by Mascot for allsamples.

Several other proteins were also detected (e.g., vitronectin, lumican,biglycan). For all samples, 7.3±2.4 proteins, 939.1±185.8 totalpeptides, and 128.4±19.1 unique peptides were detected using thisextraction and mass spectrometry method. After using proteaseinhibitors, 9 proteins were identified for the 48M sample whereas only 5were identified in the non-inhibited sample.

The 400 mM ammonium phosphate dibasic/200 mM ammonium bicarbonate/4MGuHCl extraction resulted in the greatest number of proteinidentifications (as many at 20 unique accession numbers). Collagen Ialpha 1 and 2 and osteocalcin were the highest scoring proteins for thisextraction, consistent with the other 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate extraction. Other matrix proteins(e.g., lumican, biglycan, collagen III, vitronectin, osteomodulin) werealso detected.

A number of useful conclusions may be drawn from the results reported inthe example above. First, incubation for as little as four hours usingeither of the extraction buffers (400 mM ammonium phosphate dibasic or400 mM ammonium phosphate dibasic/200 mM ammonium bicarbonate) resultedin complete protein yields. That is, as can be seen in FIG. 1 , longerincubation times (eight hours or 24 hours) did not increase proteinyield using either buffer.

Second, increased temperature alone may be sufficient, in some cases, toachieve acceptable protein yields. Referring to FIG. 2 again, it can beseen that while the 400 mM ammonium phosphate dibasic/200 mM ammoniumbicarbonate buffer was consistent in achieving higher yields than the400 mM ammonium phosphate dibasic buffer at both room temperature and at75° C., the 400 mM ammonium phosphate dibasic buffer at 75° C. had ahigher yield than did the 400 mM ammonium phosphate dibasic/200 mMammonium bicarbonate at room temperature.

Third, the addition of 200 mM ammonium bicarbonate to the extractionbuffer significantly increased protein yield over the use of 400 mMammonium phosphate dibasic alone. This result was consistent regardlessof the temperature at which the bone sample/extraction buffer wasincubated, as shown in FIG. 3 , or the age of the sample donor, as shownin FIG. 4 .

Fourth, the proteins extracted were different for each of these twobuffers. Table 1 below shows the proteins detected from the bone sampleof the 48 year-old male for all temperatures and incubation times usingthe 400 mM ammonium phosphate dibasic buffer and the 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate buffer.

TABLE 1 400 mM ammonium 400 mM ammonium phosphate dibasic/ phosphatedibasic 200 mM ammonium bicarbonate Actin, aortic smooth muscleCeruloplasmin Apolipoprotein A-I Collagen alpha-1(I) chain Collagenalpha-1(I) chain Collagen alpha-2(I) chain Collagen alpha-2(I) chainFibrinogen gamma chain Fibrinogen gamma chain Hemoglobin subunit alphaHemoglobin subunit beta Hemoglobin subunit beta Histone H2A OsteocalcinSerum albumin Vimentin Vimentin

Together, these results demonstrate the ability to tailor both theprotein yields and protein types to be extracted by varying thecomposition of the extraction buffer, the incubation temperature, orboth.

It must also be recognized that the addition of a denaturing agent (4 Mguanidine HCl) did significantly increase the total protein yield overthe 400 mM ammonium phosphate/200 mM ammonium bicarbonate alone, asshown in FIG. 3 . This also resulted in the extraction of a number ofproteins (lumican, biglycan, collagen III, vitronectin, andosteomodulin) that were not extracted using either 400 mM ammoniumphosphate alone or 400 mM ammonium phosphate/200 mM ammonium bicarbonatealone. This is a result of an increase in the solubility of matrixproteins that may not be soluble without denaturation and suggests anadditional degree of flexibility in tailoring the protein yields andtypes extracted according to embodiments of the invention.

Osteocalcin and osteomodulin were the only mineral specific proteinsdetected using the methods and buffers of the invention. However, thisresult suggests that these methods and buffers can interact with thehydroxyapatite surface sufficiently to dissociate mineral proteins.Osteocalcin was only detected consistently in extractions that employedammonium bicarbonate, suggesting that bicarbonate can disrupt thecarboxyl interaction between osteocalcin and the mineral surface.

The addition of a protease inhibitor allowed for the extraction andidentification of additional proteins (e.g., Collagen alpha-1 (XXVIII)chain). This may be critical to the wider characterization of the boneproteome. Protease inhibitors suitable for use in accordance withembodiments of the invention include, for example, sodium fluoride,sodium orthovanadate, sodium pyrophosphage, beta-glycerophosphate, andmixtures thereof.

Table 2 below summarizes some of the proteins extracted using each offour extraction buffers according to various embodiments of theinvention. In Table 2, Buffer A comprises 400 mM ammonium phosphatedibasic/200 mM ammonium bicarbonate, Buffer B comprises 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate/4 M guanidine HCl, BufferC comprises 400 mM ammonium phosphate dibasic/200 mM ammoniumbicarbonate/10 μg/mL Halt™ protease inhibitor, and Buffer D is 400 mMammonium phosphate dibasic/200 mM ammonium bicarbonate/4 M guanidineHCl/10 μg/mL Halt™ protease inhibitor.

TABLE 2 Buffer Buffer Buffer Buffer Protein A B C D Alpha-1-antitrypsinX X Alpha-2-macroglobulin X Basement membrane-specific X heparan sulfateproteoglycan core protein Biglycan X X X Chondroadherin X X X Collagenalpha-1(I) chain X X X X Collagen alpha-1(III) chain X X Collagenalpha-1 (V) chain X Collagen alpha-1 (V) chain/ X Collagen alpha-1 (IX)chain Collagen alpha-1 (V) chain/ X Collagen alpha-2 (XI)/ Collagenalpha-1 (XI) chain Collagen alpha-1(XI) chain X Collagen alpha-1(XXVIII) X chain Collagen alpha-2(I) chain X X X Collagen alpha-2(VI)chain X Collagen alpha-2 (XI) chain X Collagen alpha-3(VI) chain XCollagen alpha-5(IV) chain X X Glyceraldehyde-3-phosphate Xdehydrogenase Hemoglobin subunit beta X X Histone H2A 1-B/E X X Iggamma-1 chain C region X X Ig kappa chain C region X X XKeratin-associated protein 1-1 X Lumican X X X X Myoglobin X Myosinlight chain ⅓, X skeletal muscle isoform Osteocalcin X X X XOsteomodulin X Pigment epithelium-derived X factor Serum albumin X X X XTenascin X Vimentin X Vitronectin X X X Zinc finger protein 197 X

Applicant further found that the volume of extraction buffer for a givenmass of bone sample affected the total protein yield. For example, FIG.5 shows the protein yields for four extractions of bone samples of thesame mass and source. In FIG. 5 , Buffer B comprises 400 mM ammoniumphosphate dibasic/200 mM ammonium bicarbonate/4 M guanidine HCl andBuffer B-1 comprises 1 M ammonium phosphate dibasic/200 mM ammoniumbicarbonate/4 M guanidine HCl. As can be seen in FIG. 5 , the increasedconcentration of ammonium phosphate dibasic increased total proteinyield using both 600 μL and 1 mL of extraction buffer. At the same time,the 1 mL extractions with both buffers had higher total protein yieldsthan did their 600 μL counterparts.

Protein concentration was also found to increase with increasing mass ofthe bone sample extracted. FIG. 6 shows a plot of protein concentration(kg/mL) as a function of bone mass (mg) for a total of 34 extractionsusing extraction buffers according to embodiments of the invention.Although not linear, the increase in protein concentration is steadythrough bone masses of about 90 mg. This suggests that the extractionmethods of the invention are effective across a wide range of samplemass.

It was also found that extraction buffers employing other cations weresimilarly effective in terms of protein yield. FIG. 7 , for example,shows the plots of protein yields of six extraction buffers according tothe invention as compared to both 4 M guanidine HCl (GuHCl) alone andhydrochloric acid (HCl) alone. HCl is a commonly-used demineralizationagent. All extractions were of 60 mg bone samples from the same source.All other parameters were the same among the extractions.

In FIG. 7 , Buffer E comprises 400 mM sodium phosphate dibasic/200 mMsodium bicarbonate/4 M guanidine HCl, Buffer F comprises 1 M sodiumphosphate dibasic/200 mM sodium bicarbonate/4 M guanidine HCl, Buffer Gcomprises 400 mM potassium phosphate dibasic/200 mM potassiumbicarbonate/4 M guanidine HCl, Buffer H comprises 1 M potassiumphosphate dibasic/200 mM potassium bicarbonate/4 M guanidine HCl, andBuffers B and B-1 are as described above with respect to FIG. 5 .

As can be seen sodium phosphate dibasic/sodium bicarbonate buffers andpotassium phosphate dibasic/potassium bicarbonate buffers resulted inprotein yields similar to the ammonium phosphate dibasic/ammoniumbicarbonate buffers described above. Guanidine HCl alone and HCl aloneresulted in the lowest protein yields.

The types of proteins extracted using other cation-based buffers weresimilar to those using the ammonium-based buffers described above. Table3 below summarizes some of the proteins extracted using four differentbuffers according to embodiments of the invention. In Table 3, Buffer Fand Buffer H are as described above with respect to FIG. 7 , GuHCl isguanidine HCl alone, and Buffer B-2 comprises 1 M ammonium phosphatedibasic/200 mM ammonium bicarbonate/4 M guanidine HCl/0.015 Mphenacylthiazolium bromide (PTB). PTB is reagent that can help enhanceprotein recovery by breaking advanced glycation end-product crosslinks.

TABLE 3 Buffer Buffer Buffer Protein E H GuHCl B-2 Alpha-1-acidglycoprotein 1 X Alpha-2-HS-glycoprotein X X X Androgen receptor XBiglycan X X X Cathepsin K X Chondroadherin X X Collagen alpha-1(I)chain X X X Collagen alpha-1(II) chain X X X Collagen alpha-2(I) chain XX X Decorin X Dermatopontin X Fibrinogen gamma chain X Histone H2A 1-B/EX Histone H2B type 1-B X Ig gamma-1 chain C region X X Ig kappa chain Cregion X X Ig lambda chain C region X X X Lumican X X X Osteocalcin X XX Pigment epithelium-derived factor X X X Prothrombin X X X Serumalbumin X X X Vitronectin X X X

As can be seen in Table 3, Buffer B-2, containing PTB, is the onlybuffer tested that enabled the extraction of cathepsin K. Guanidine HClalone enabled the extraction of several proteins that the other buffersdid not, although the overall protein yield was lower, as shown in FIG.7 .

The addition of PTB to extraction buffers of the invention results in anincrease in total protein yield, particularly with increased incubation,even at a low temperature. FIG. 8 , for example, shows plots of totalprotein yield for two buffers according to the invention, each after twoincubation periods. In FIG. 8 , Buffer B-3 comprises 400 mM ammoniumphosphate/200 mM ammonium bicarbonate/4 M guanidine HCl 0.015 M PTB andBuffer B-2 is as described above with respect to Table 3.

Extractions following incubation for 20 hours at 4° C. increased thetotal protein yield for both buffers, as compared to yields obtainedupon immediate extraction. In both cases, the yields for Buffer B-3 werehigher than for Buffer B-2.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any related or incorporated methods. Thepatentable scope of the invention is defined by the claims, and mayinclude other examples that occur to those skilled in the art. Suchother examples are intended to be within the scope of the claims if theyhave structural elements that do not differ from the literal language ofthe claims, or if they include equivalent structural elements withinsubstantial differences from the literal language of the claims.

Although the disclosed subject matter has been described and illustratedwith respect to embodiments thereof, it should be understood by thoseskilled in the art that features of the disclosed embodiments can becombined, rearranged, etc., to produce additional embodiments within thescope of the invention, and that various other changes, omissions, andadditions may be made therein and thereto, without parting from thespirit and scope of the present invention.

What is claimed is:
 1. A bone protein extraction buffer effective to solubilize matrix proteins from a sample of solid bone, comprising: ammonium phosphate dibasic, ammonium bicarbonate, guanidine HCl, and at least one protease inhibitor, wherein the at least one protease inhibitor is selected from the group consisting of: sodium fluoride, sodium orthovanadate, sodium pyrophosphate, beta-glycerophosphate, and mixtures thereof, wherein the bone protein extraction buffer does not include a demineralization agent, and the extraction buffer includes ammonium phosphate dibasic at a concentration of 400 mM.
 2. The extraction buffer of claim 1, wherein the extraction buffer includes ammonium bicarbonate at a concentration of about 200 mM.
 3. The extraction buffer of claim 1, wherein the extraction buffer further includes guanidine HCl at a concentration of about 4 M.
 4. The extraction buffer of claim 1, further comprising: phenacylthiazolium bromide (PTB). 